Ray, P., De, S., Mitra, A., Bezstarosti, K., Demmers, J. A., Pfeifer, K. and Kassis, J. A. (2016). Combgap contributes to recruitment of Polycomb group proteins in Drosophila. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 27001825Summary:Polycomb group (PcG) proteins are responsible for maintaining the silenced transcriptional state of many developmentally regulated genes. PcG proteins are organized into multiprotein complexes that are recruited to DNA via cis-acting elements known as "Polycomb response elements" (PREs). In Drosophila, PREs consist of binding sites for many different DNA-binding proteins, some known and others unknown. Identification of these DNA-binding proteins is crucial to understanding the mechanism of PcG recruitment to PREs. This study reports the identification of Combgap (Cg), a sequence-specific DNA-binding protein that is involved in recruitment of PcG proteins. Cg can bind directly to PREs via GTGT motifs and colocalizes with the PcG proteins Pleiohomeotic (Pho) and Polyhomeotic (Ph) at the majority of PREs in the genome. In addition, Cg colocalizes with Ph at a number of targets independent of Pho. Loss of Cg leads to decreased recruitment of Ph at only a subset of sites; some of these sites are binding sites for other Polycomb repressive complex 1 (PRC1) components, others are not. These data suggest that Cg can recruit Ph in the absence of PRC1 and illustrate the diversity and redundancy of PcG protein recruitment mechanisms.

Hitrik, A., Popliker, M., Gancz, D., Mukamel, Z., Lifshitz, A., Schwartzman, O., Tanay, A. and Gilboa, L. (2016). Combgap promotes ovarian niche development and chromatin association of EcR-binding regions in BR-C. PLoS Genet 12: e1006330. PubMed ID: 27846223Summary:
The development of niches for tissue-specific stem cells is an important aspect of stem cell biology. Determination of niche size and niche numbers during organogenesis involves precise control of gene expression. How this is achieved in the context of a complex chromatin landscape is largely unknown. This study shows that the nuclear protein Combgap (Cg) supports correct ovarian niche formation in Drosophila by controlling Ecdysone-Receptor (EcR)- mediated transcription and long-range chromatin contacts in the broad locus (BR-C). Both cg and BR-C promote ovarian growth and the development of niches for germ line stem cells. BR-C levels were lower when Combgap was either reduced or over-expressed, indicating an intricate regulation of the BR-C locus by Combgap. Polytene chromosome stains showed that Cg co-localizes with EcR, the major regulator of BR-C, at the BR-C locus and that EcR binding to chromatin was sensitive to changes in Cg levels. Proximity ligation assay indicated that the two proteins could reside in the same complex. Finally, chromatin conformation analysis revealed that EcR-bound regions within BR-C, which span ~30 KBs, contacted each other. Significantly, these contacts were stabilized in an ecdysone- and Combgap-dependent manner. Together, these results highlight Combgap as a novel regulator of chromatin structure that promotes transcription of ecdysone target genes and ovarian niche formation.

Davis, T. L. and Rebay, I. (2017). Antagonistic regulation of the second mitotic wave by Eyes absent-Sine oculis and Combgap coordinates proliferation and specification in the Drosophila retina. Development 144(14):2640-2651. PubMed ID: 28619818Summary:
The transition from proliferation to specification is fundamental to the development of appropriately patterned tissues. In the developing Drosophila eye, Eyes absent (Eya) and Sine oculis (So) orchestrate the progression of progenitor cells from asynchronous cell division to G1 arrest and neuronal specification at the morphogenetic furrow. This study uncovered a novel role for Eya and So in promoting cell cycle exit in the Second Mitotic Wave (SMW), a synchronized, terminal cell division that occurs several hours after passage of the furrow. Combgap (Cg), a zinc-finger transcription factor, antagonizes Eya-So function in the SMW. Based on Cg's ability to attenuate Eya-So transcriptional output in vivo and in cultured cells and on meta-analysis of their chromatin occupancy profiles, it is speculated that Cg limits Eya-So activation of select target genes posterior to the furrow to ensure properly timed mitotic exit. This work supports a model in which context-specific modulation of transcriptional activity enables Eya and So to promote both entry into and exit from the cell cycle in a distinct spatiotemporal sequence.

The combgap (cg) locus, first described by C. B. Bridges in 1925, is a gene required for proper anteroposterior pattern formation in the limbs of Drosophila. combgap mutation has pleiotropic effects on bristle number (most notably for the male sex comb), wing venation, and oogenesis. Based on genetic interactions and phenotypic analysis, cg was assigned to the 'vein' group of loci that function in wing vein patterning (Song, 2000 and references therein). combgap encodes a chromosomal protein with 11 C2H2 zinc fingers. Limb defects found in combgap mutants consist of either loss or duplication of pattern elements in the anteroposterior axis and can be explained through the inappropriate expression of cubitus interruptus (ci) and its downstream target genes (Campbell, 2000; Svendsen, 2000; Song, 2000). A genetic interaction between cg and some ci mutants was identified many years ago (House, 1953, 1961; Waddington, 1953). In cg mutants, ci is ectopically expressed in the posterior compartments of wing imaginal discs and is downregulated in the anterior compartment of legs, wings and antennae. Combgap protein binds to polytene chromosomes at many sites including the ci locus, suggesting that it could be a direct regulator of ci transcription. The adult viable cg mutants are sterile and also exhibit defects such as eye roughening and ectopic thoracic bristles. Several deficiencies from the Bloomington Deficiency Collection have dominant phenotypes (i.e., wing notching, ectopic veins) when in compound heterozygotes with cg2. While some of these phenotypes may be due to effects on ci regulation, others are not easily ascribed to changes in ci expression. Thus, cg may be required for the regulation of many genes but ci may be particularly sensitive to reduction in cg function (Svendsen, 2000).

Transcriptional control of the ci gene and post-translational regulation of the Ci protein are essential for the placement and function of the A/P organizer in limb development. The action of the Hedgehog-signaling pathway results in a relatively high ratio of Ci-155 (the activator form of Ci) to Ci-75 (the repressive form of Ci) at the A/P boundary, which ensures the localized expression of Hh-responsive genes necessary for the formation of the A/P organizer. Hh signaling influences the post-translational modification of the Ci protein at multiple levels including proteolytic cleavage, phosphorylation, subcellular location and nuclear import. In contrast to the details of post-translational modification of Ci-155 by elements of the Hh-signaling pathway, relatively little is known about the transcriptional control of ci. Decreased levels of both Ci protein and expression from ci-lacZ reporter constructs are observed in the anterior compartments of cg mutant imaginal discs, as well as ectopic expression in posterior cells of wings. Thus, Cg affects both the activation and repression of ci transcription (Svendsen, 2000).

A dominant interaction between cg and engrailed/invected mutations that gives rise to a gap in vein L4 strongly suggests that Cg and En/Inv act together to repress posterior ci transcription. Posterior expression of En represses the transcription of ci resulting in anterior specific expression. En has been shown to interact directly with the ci regulatory elements. In cg mutant wing imaginal discs, weak ectopic expression of ci-lacZ reporter constructs are found in posterior cells, thus Cg may act in concert with En to repress posterior ci. Hypomorphic mutants in either cg or en/inv can give rise to the reduction in vein L4 that is characteristic of ectopic ci expression (Svendsen, 2000).

Many proteins with multiple C2H2 zinc finger motifs like those found in cg have been shown to be transcription factors, DNA-binding proteins or chromatin proteins. The widespread localization of Cg on salivary gland chromosomes is consistent with all of these activities. While the data have not yet established direct action of Cg on the ci regulatory elements, binding of Cg to the ci region of polytene chromosomes suggests that Cg could be a direct regulator of ci transcription. Direct binding of Cg (produced in E. coli) to DNA from the ci regulatory region has not been detected. However, given that the transcriptional regulation of ci is likely to be complex, Cg may not act at the level of direct DNA binding. The involvement of the Pc-group genes in the repression of ci suggests that intricate regulatory modes are necessary to maintain the correct levels and spatial patterns of ci transcription during imaginal disc development. Furthermore, the ci-regulatory regions have been shown to be subject to transvection effects, indicating that interchromosomal interactions also govern ci regulation. Thus Cg may act at any level, from generally influencing the chromosome pairing through to direct binding of ci enhancer elements. Finally, the positive and negative effects of cg mutants on ci transcription and the genetic interaction with en/inv suggest that Cg may be required in conjunction with other transcription factors for the function of ci enhancers and that Cg may not specify activation or repression itself (Svendsen, 2000).

The changes in the A/P pattern observed in cg mutant limbs are caused by the mis-regulation of Hh-responsive genes regulated by the Ci-155 and Ci-75 transcription factors. In cg mutant wing imaginal discs, Ci-155 is ectopically expressed in the posterior compartment and is associated with posterior compartment defects and posterior misexpression of genes such as patched and knot. Ectopic expression of kn is sufficient to suppress vein fate. Thus, the misexpression in the posterior compartment of kn and other genes regulated by high levels of Ci-155 probably leads to the vein defects described in this study. The occurrence of both higher levels of posterior Ci and Kn expression, and higher frequency of posterior compartment defects in cg1/cg1 mutant wings supports this explanation. Stronger allelic combinations of cg have lower levels of ectopic Ci and Kn, and lower incidence of posterior compartment wing defects, but they result in a greater reduction in Ci in the anterior compartment and more anterior vein defects. The posterior and anterior vein defects, as well as occasional anterior wing margin bifurcations, resemble the effects of regulatory mutants of ci that cause the ectopic expression of ci in posterior cells and the reduction of ci expression in the anterior compartment (Svendsen, 2000 and references therein).

In legs and antennae, overall Ci levels are decreased in the anterior compartment, resulting in circumferential overgrowth of the anterior compartment and ectopic anterior expression of the morphogens wg and dpp. Similar effects on leg morphology have been previously reported when wg and dpp were ectopically activated in anterior cells. The rescue of cg mutant leg defects by additional expression of ci in the anterior compartment using the Gal4/UAS system indicates that the phenotypes result from a reduction of Ci-75 leading to the derepression of wg and dpp. Thus, it is concluded that the Cg protein is critical for the proper levels and spatial patterns of Ci and that the A/P limb patterning defects in cg mutants are due largely, if not completely, to mis-regulation of ci (Svendsen, 2000).

The effects of cg mutants on ci expression are seen only in the anterior compartment of legs but in both anterior and posterior compartments in wings. What is the basis for this difference? While anterior Ci is reduced in both limbs, ectopic posterior Ci is only seen in wings. One possibility is that the alleles that have been studied may have different effects on cg expression in anterior versus posterior compartments and/or legs versus wings. However, little Cg imaginal disc staining was seen in cg2/cg2, suggesting little or no Cg protein is produced, and so the phenotype may be near null (there are no deficiencies uncovering the cg locus, so this could not be tested genetically). Cg may not be required for repression of ci in the posterior compartment of leg discs, or alternatively, there may be a much lower threshold for Cg function in legs. The different effects on ci expression in cg mutant leg and wing imaginal discs suggest that while the broad framework is similar, there may be unique aspects to A/P patterning in dorsal versus ventral limbs. The predominance of wing phenotypes in ciW and similar ci regulatory mutations also suggests a difference in the way ci is regulated in wings versus legs. Another difference was seen in the effects of reduced Ci levels on the expression of dpp. A greater reduction of Ci staining is seen in the anterior compartments of wings compared with leg imaginal discs; paradoxically, ectopic expression of dpp is seen in all cg mutant leg imaginal discs but none is seen in cg2/cg2 wing imaginal discs. Although there are limits to how accurately real levels of Ci may be inferred from histochemical staining in different tissues, the simple conclusion is that dpp responds to different thresholds of Ci in legs and wings, and that the effect of cg is indirect (Svendsen, 2000).

Similar conclusions about the role of cg in ci regulation have been reached in the Campbell (2000) study. The posterior wing venation defect in cg hypomorphs is very similar to that found in ci mutants and this phenotype is enhanced in cg/+;ci/+ transheterozygotes. These ci mutants, however, are gain-of-function mutants; they show ectopic expression of ci in the posterior. In fact, direct misexpression of ci in the posterior using the UAS/Gal4 system can also produce the same vein defects as seen in these mutants and in cg mutants. Analysis of cg mutant discs reveals ectopic ci expression in the posterior, indicating that the cg posterior phenotype is almost certainly the direct result of deregulation of ci expression in this compartment (Campbell, 2000).

Ci expression is also abnormal in the anterior of cg mutant discs, being found at much lower levels than in wild-type discs. Loss of ci expression in the wing results in hedgehog gain-of-function phenotypes, including overgrowth and misexpression of dpp. Reduced Ci levels in the leg also result in the characteristic overgrowth phenotype, with ectopic expression of wg and dpp, found following ubiquitous expression of Hh -- i.e., the same phenotype as that found in cg mutant leg discs. Support for the proposal that the anterior combgap phenotype in the leg is also the direct result of deregulation of ci expression, in this case lowered levels of expression, comes from the observation that raising ci levels in cg mutant leg discs using the UAS/Gal4 system can suppress the overgrowth and ectopic dpp expression (Campbell, 2000).

One difference between ci and cg mutants is that wing discs from the former have a hedgehog gain-of-function phenotype with overgrowth and ectopic dpp in the anterior, while the latter do not show overgrowth and only very weak ectopic dpp. It is possible that the leg and wing are differentially sensitive to Ci levels and the Ci levels are still high enough in the wing in cg mutants to repress most dpp expression. Protein levels detected with antibody staining in ci hypomorphs and cg mutants are too low to detect significant differences with confidence, so the reason for the difference between ci and cg wings remains to be determined. Ci is also required during embryogenesis, but the putative null cg mutant survives to the early pupal stage. This suggests either that lower levels of Ci are sufficient for embryonic but not larval development or that cg RNA is maternally supplied. The first possibility is supported by the observation that hypomorphic ci mutants are not embryonic lethal and survive to the early pupal stage. However, in situ analysis reveals that CG RNA is maternally supplied so that the question of whether cg is required during embryogenesis will require the generation of germline clones (Campbell, 2000).

Full-length Ci acts as a transcriptional activator and there is evidence that the lowered levels of Ci in cg mutants also compromises Ci function as an activator. Although, dpp is misexpressed in cg discs, the level of expression, even at the compartment border, is lower than that found in wild-type discs. A similar phenomenon has been demonstrated for loss of ci in the wing and it appears that the high levels of dpp in wild-type discs require activation by Ci-155, as well as the absence of Ci-75. Thus, the lower levels of dpp in cg discs are presumably due to lower levels of Ci-155. Another gene directly activated by Ci is en in late third instar wing discs. Ci-dependent en activation in the anterior compartment does not occur in cg mutant cells, again presumably because the level of the Ci-155 activator form is too low (Campbell, 2000).

Thus Cg is required to activate ci expression to its normal levels in the anterior compartment and to repress ci expression in the posterior. The Cg protein contains multiple zinc fingers and is most probably a DNA-binding protein that would be expected to bind to elements at the ci locus. However, understanding the mechanism by which it regulates ci expression requires further studies. It is possible that Cg functions as a standard transcription factor and activates ci transcription in the anterior and represses it in the posterior. If this is the case, its activity must be modified in either the anterior or posterior compartments. Analysis of the Cg protein outside of the zinc fingers does not reveal any classical activator or repressor domains, but as these are often not well defined it is impossible to determine whether the protein has these activities without more-detailed studies (Campbell, 2000).

An argument against such a direct involvement of Cg in transcription is the well-documented role of En in regulating ci expression. En is a transcription factor that represses expression of several genes including ci, dpp and wg, and has been shown to bind to elements at the ci locus. It would appear likely that En is the primary factor that represses transcription of ci in the posterior. If this is the case, the function of Cg in regulating transcription may be indirect and may be to assist the binding of other transcription factors to the ci gene. If so, the misexpression of Ci in the posterior of cg mutant discs would be due to a lowered ability of En to bind in the absence of Cg protein, while the lowered Ci levels in the anterior would be due to a lowered ability to bind a currently unidentified transcriptional activator of ci. There are several possible mechanisms by which Cg might affect the binding of other factors. For example, there may be direct physical interactions between Cg and these other factors. Alternatively, Cg action could be more indirect, for example, it could modify chromatin structure at the ci locus producing a more open conformation. Further studies are required to test these possibilities (Campbell, 2000).

The morphogenesis of specialized structures within the CNS relies on the nonautonomous activity of cell populations that play the role of organizers. In the Drosophila visual system, cells on the dorsal and ventral margins of the developing visual cortex express the Wnt family member Wingless (Wg) and the TGF-beta Decapentaplegic (Dpp). The activity of these morphogens in establishing cortical cell fates sets the stage for the guidance of photoreceptor axons to their retinotopic destinations in the Drosophila brain. One role for Wg in cortical development is to induce and maintain the expression of Dpp, a key step in the assignment of dorsoventral cell identities. Dpp is induced early in cortical development, shortly after the onset of Wg expression in a few dorsal and ventral margin cells, and is maintained by Wg activity until at least the time of retinal axon pathfinding. Wg is a developmental signal in many different tissues, and acts by regulating different target gene sets to elicit a constellation of different cell fates. Wingless-controlled targets include distal-less and vestigial in the wing, engrailed in the embryonic ectoderm, labial in the gut, and sloppy-paired in the embryonic CNS. Conversely, Dpp belongs to a Hedgehog-controlled circuit in the wing (Song, 2000 and references therein).

A regulatory mechanism is described that relays Wg signal reception to the tissue-specific expression of target genes in the visual cortex. In a screen for mutants in which photoreceptor axons project aberrantly to their destinations in the brain, a mutation in combgap was discovered. Retinal axon navigation defects in combgap animals are due to the role of cg in the establishment of cortical cell identity. cg represses the expression of Wg target genes in a positionally restricted manner in the visual cortex. wg+ induction of its cortical cell targets occurs via the downregulation of cg. Combgap is thus a tissue-specific relay between Wingless and its target genes for the determination of cell fate in the visual cortex (Song, 2000).

The translation of extracellular signals into cell-type identities is an important step in the elaboration of complex patterns of neuronal connectivity. In the visual system of Drosophila, a population of about 40 cells set aside in embryogenesis gives rise to thousands of cortical neurons that form three precisely interconnected visual ganglia. Hundreds of neuronal and glial cell types are generated, with more than 120 distinct neuronal cell types identified in the medulla cortex alone. Wingless plays an important role in the determination of visual system cortical cell fates, acting from cell populations at the dorsal and ventral margins to induce expression of the morphogen Dpp and the transcriptional regulators Distal-less. combgap is a key regulator in this pathway for cortical cell fate determination (Song, 2000).

A combgap mutation was recovered in a screen for mutants with aberrations in retinal axon projections. On the basis of its effects on target region gene expression and the outcome of mosaic analysis, it is evident that a role for combgap in the specification of cortical cell identity underlies its requirement for the establishment of retinotopic connectivity in the visual system. In cg loss of function animals, three markers under wg+ control are expressed in expanded dorsal and ventral portions of the retinal axon target field. The requirement for cg to repress the markers within these domains is autonomous. The lamina midline region, however, appears phenotypically normal in homozygous or mosaic cg animals. This positionally restricted requirement for cg+ activity is correlated with the pattern of cg expression, since cg is not expressed in the midline region where it is not required. Since wg+ misexpression is sufficient to induce wg+-dependent markers in the midline region, another regulatory system must control these markers there. Hence, the consequences of wg signal reception at different dorsoventral positions within the cortical precursor field would appear to involve a set of regulatory molecules that divide the cortex into specific domains for pattern formation (Song, 2000).

As in the wing and leg, cg clones that include the cortical lamina are deficient in Ci expression. These effects on Ci expression could account for the reduction of the Hh-controlled expression of Engrailed and Dpp at the anterior-posterior compartment border of the developing wing. However, Ci loss and gain of function experiments have not revealed a role for Ci in the regulation of Wg target gene expression in visual system cortical cells where Ci functions as in other imaginal tissues as an effector of Hedgehog signal reception. Moreover, the cortical markers under Wg control do not respond to Hh (Song, 2000).

The constellation of genes under Wingless control displays considerable tissue specificity. Wingless-controlled targets include Distal-less and vestigial in the wing, engrailed in the embryonic ectoderm, and sloppy-paired in the embryonic CNS. Though Dpp and Omb belong to a Hedgehog-controlled circuit in the wing, they are under Wg control in the visual cortices of the brain. With respect to the control of cell fate, Wg signal transduction apparently follows a canonical pathway from a pair of redundant receptors at the cell surface to the cytoplasmic control of Armadillo stability and nuclear translocation. This raises the question of how the tissue specificity of wg target gene expression is achieved (Song, 2000).

The observations that cg regulates dpp, optimotor blind and aristaless in the visual cortex place cg in a second tier of regulation, as a component of a tissue-specific relay mechanism between the Wg signal transduction pathway and the target genes that are wg dependent in visual system cortical cells. The evidence in support of this hypothesis is as follows: (1) epistasis analysis with the wg pathway negative regulator Axn places the requirement for cg downstream of the cytoplasmic complex that includes APC, GSK-beta, and Armadillo; (2) the induction of at least three downstream effectors of wg+ activity is mediated by negative regulation of cg expression -- cg expression is reduced in the dorsal and ventral domains of the cortical lamina where these wg target genes are expressed and ectopic cg expression blocks wg target gene expression within these domains; (3) ectopic wg+ clones repress cg expression, yielding Cg-negative domains in which wg target genes are ectopically expressed. The presence of consensus Pangolin binding sites in the first intron of cg suggests cg may be a direct target of Wg signal transduction. How the Armadillo/Pangolin complex might participate in the negative regulation of cg is unclear. Cg might act by binding directly to wg target gene regulatory elements as a transcriptional repressor (Song, 2000).

Additional molecules have been identified as tissue- and/or stage-specific modulators of wg signal transduction. The lines gene product is specifically required for late stage Wg signaling in the dorsal epidermis of the embryo. Wg regulates the nuclear accumulation of Lines rather than its expression. Teashirt, a zinc finger protein, modulates Wg signaling specifically in the ventral epidermis by binding to the C terminus of Arm. In addition, Smad transcriptional effectors of TGF-beta signaling can bind the Arm/dTCF complex to mediate tissue-specific cross-regulatory interactions between the TGF-beta and Wnt pathways. Along with the mode of action of Cg in the visual cortex, the limited number of examples so far point to a diversity of mechanisms for achieving the tissue-specific consequences of Wnt signaling (Song, 2000 and references therein).

combgap is identified in a screen for genes that function in leg disc regeneration in Drosophila

Many diverse animal species regenerate parts of an organ or tissue after injury. However, the molecules responsible for the regenerative growth remain largely unknown. The screen reported in this study aimed to identify genes that function in regeneration and the transdetermination events closely associated with imaginal disc regeneration using Drosophila melanogaster. A collection of 97 recessive lethal P-lacZ enhancer trap lines were screened for two primary criteria: first, the ability to dominantly modify wg-induced leg-to-wing transdetermination and second, for the activation or repression of the lacZ reporter gene in the blastema during disc regeneration. Of the 97 P-lacZ lines, six genes (Krüppelhomolog- 1, rpd3, jing, combgap, Aly and S6 kinase) were identified that met both criteria. Five of these genes suppress, while one enhances, leg-to-wing transdetermination and therefore affects disc regeneration. Two of the genes, jing and rpd3, function in concert with chromatin remodeling proteins of the Polycomb Group (PcG) and trithorax Group (trxG) genes during Drosophila development, thus linking chromatin remodeling with the process of regeneration (McClure, 2008).

There are three different mechanisms that organisms use to re-grow and replace lost or damaged body parts, and often, more than one mechanism can function within different tissues of the same organism. Muscle and bone, for example, repair themselves by activating a resident stem cell population, while the liver regenerates by compensatory proliferation of normally quiescent differentiated cells. Appendage/fin regeneration in lower vertebrates occurs by a process termed epimorphic regeneration, which proceeds in three distinct stages: (1) wound healing and migration of the surrounding epithelial cells to form the wound epidermis, (2) formation of the regeneration blastema -- a mass of undifferentiated and proliferating cells of mesenchymal origin and (3) regenerative outgrowth and pattern re-formation. Whether these diverse modes of regeneration share a common molecular and genetic basis is not known (McClure, 2008).

Regeneration in the Drosophila imaginal discs, the primordia of the adult fly appendages, closely parallels epimorphic limb/fin regeneration in lower vertebrates. Cells in the imaginal discs are rigidly determined to form specific adult structures (e.g., legs and wings) by the third larval instar. If the discs are fragmented at this time and cultured in vivo, they will regenerate. Disc regeneration begins 12 h after wounding, when transient heterotypic contacts are made between peripodial (squamous epithelium) and columnar cells (disc proper) near the cut edges of the wound. These initial contacts involve microvilli-like extensions and provide temporary wound closure. Then, approximately 24 h after wounding, homotypic cell contacts (between columnar or between squamous cells) are made involving the close apposition of cell membranes and cellular bridges, which eventually (48 h after wounding) restore the physical continuity of the disc. Before and during wound healing, cell division is randomly distributed throughout the disc. However, once completed (36-48 h after wounding), division is observed only in cells near the wound site. These cells are known as the regeneration blastema. Thus, like appendage regeneration in lower vertebrates, disc regeneration involves wound healing followed by blastema formation (McClure, 2008).

Blastema cells are responsible for the regeneration and repatterning of the entire missing disc fragment. Thus, these cells exhibit remarkable developmental plasticity. For example, in anterior- only leg disc fragments, some blastema cells will switch to posterior identity and establish a novel posterior compartment in the regenerate. This anterior/posterior conversion occurs during heterotypic wound healing, when hedgehog (hh)- expressing peripodial cells induce ectopic engrailed (en) expression in the apposing anterior columnar cells. In addition, the disc blastema, like its vertebrate counterpart, is able to form a normal regenerate (complete leg disc and adult leg) when isolated from the remaining disc fragment. Regenerative plasticity is also observed when a few blastema cells switch fate to that of another disc type (e.g., leg-to-wing), in a phenomenon known as transdetermination. Transdetermination events are closely associated with regenerative disc growth. Clonal analysis, for example, has shown that blastema cells first regenerate the missing disc structures, and only then, are they competent to transdetermine (McClure, 2008).

Little is known about how the regeneration blastema forms in the fragmented leg disc, although ectopic Wingless (Wg/Wnt1) expression is detected along the cut site, both prior to and during blastema formation. Wg is a developmental signal in many different tissues and animals; in flies Wg patterns all of the imaginal discs, functioning as both a morphogen and mitogen to regulate disc cell fate and growth. In lower vertebrates, Wnt ligands are key regulators of blastema formation during epimorphic regeneration. Thus, activation of Wg within the disc blastema is potentially important for regeneration. This idea is consistent with the observation that ubiquitous expression of wg during the second or third larval instars, in unfragmented leg discs, is sufficient to induce a regeneration blastema in the proximodorsal region of the disc, known as the weak point. Moreover, ubiquitous expression of wg mimics the pattern deviations associated with leg disc fragmentation and subsequent regeneration, including the duplication of ventral with concomitant loss of dorsal pattern elements and leg-to-wing transdetermination events. Thus, leg disc regeneration can be examined using two experimental protocols: fragmentation or ubiquitous wg expression. However, it is important to point out that only fragmentation-induced regeneration involves wound healing (McClure, 2008).

Precisely which molecules and signaling pathways are required for the process of regeneration remain poorly understood, partly because the organisms historically used to study regeneration (e.g., newts and salamanders) have been refractory to genetics and molecular manipulations. Recently, however, the use of new genetic techniques together with 'regeneration' model systems -- such as planarians, hydra and zebrafish have given researchers the opportunity to examine the mechanisms of regeneration and to identify the genes, proteins and signaling pathways that regulate different regenerative processes. For example, a large scale RNAi-based screen was performed to survey gene function in planarian tissue homeostasis and regeneration. Out of ~1000 genes examined, RNAi knock-down of 240 displayed regeneration-related phenotypes, including defects in wound healing, blastema formation and blastema cell differentiation. Despite these studies, however, it remains unclear whether regeneration requires only the modulation of genes expressed at the time of injury, the reactivation of earlier developmental genes and/or signaling pathways, or the activation of novel genes specific to the process of regeneration. Thus, a major interest in the field of regenerative biology is the identification of gene products that regulate blastema formation, blastema growth and regenerative cellular plasticity. A genetic screen, using wg-induced leg disc regeneration, aimed at identifying genes that regulate cellular plasticity and regeneration using Drosophila was carried out prothoracic leg discs. A collection of 97 recessive lethal P-element lacZ (PZ) insertion lines were screened for ectopic lacZ expression during wg-induced leg disc regeneration, and six genes were identified that function in wg-induced leg disc regeneration, including genes with functional ties to Wg signaling as well as chromatin remodeling proteins (McClure, 2008).

This study consisted of an enhancer trap screen designed to identify genes with changed gene expression during leg disc regeneration as well as required for regenerative proliferation and growth. The screen identified 19 genes that when heterozygous mutant (PZ/+), dominantly modify wg-induced leg-to-wing transdetermination, which serves as a functional assay for disc regeneration. Of the 19 genes, 37% are transcription factors or involved in transcriptional regulation (tai, Krh1, ken, jing, combgap (cg), rpd3 and Aly), 21% function in cell cycle regulation and growth (oho23B, S6k, polo and cycA), 10.5% play a role in protein secretion (Secβ61 and Syx13), and 31% are of other or unknown function [l(3)01629, CG30947, l(2)00248, l(3)05203, l(3)01344, Nup154]. The identification of transcription factors as the most frequent class of genes that modify wg-induced leg disc regeneration was similarly observed in a DNA microarray screen designed to identify genes enriched in leg disc cells that transdetermine to wing (Klebes, 2005). Together, these findings strongly suggest that transcription factors and their downstream targets play a prominent role in disc cell plasticity (McClure, 2008).

Using lacZ expression analyses, together with whole mount in situ hybridization experiments, the expression patterns of the 19 genes that modified wg-induced leg-to-wing transdetermination were verified. This analysis identified several different expression patterns upon wg-induced regeneration, including a loss of gene expression, ubiquitous expression and genes with expression limited to the regeneration blastema. Such observations indicate that a complex change of gene expression, both negative and positive, mediates the process of epimorphic regeneration. Six (jing, Alyi cg, rpd3, Kr-h1 and S6k) of the 19 modifiers displayed expression limited to the regeneration blastema, indicating that novel markers of regeneration and transdetermination have been identified. The blastema-specific expression patterns of jing, Aly, cg, Kr-h1, rpd3 and S6k raised the intriguing possibility that these genes may be functionally involved in the formation, cell proliferation or maintenance of the blastema during disc regeneration. Indeed, upon ubiquitous wg expression jing/+ animals rarely formed a regeneration blastema, indicating that two wild-type copies of jing are required for the initiation of the regenerative process. In contrast, Aly/+ and cg/+ animals formed a normal blastema, but only after a one-day delay. Therefore, two wild-type copies of the Aly and cg genes are required for the proper timing of regeneration. In addition, it was found that the frequency of blastema formation was reduced in rpd3/+ animals, implicating this gene in the process of regeneration. Interestingly, heterozygous mutations in all four of these genes (jing, Aly, cg and rpd3) strongly suppress wg-induced leg-to-wing transdetermination. It is speculated that the transdetermination frequency declines in these mutant animals because the initiation and/or timing of blastema formation is delayed. This idea is consistent with all previous work which has shown that blastema cells are only competent to transdetermine after they have regenerated the missing disc structures. Heterozygous mutations in Kr-h1 and S6k did not significantly alter the formation of the wg-induced regeneration blastema, however, these genes did affect regeneration-induced transdetermination. Such results suggest that Kr-h1 and S6k specifically function to modulate the cell fate changes that occur as a consequence of regeneration (McClure, 2008).

Investigations into the molecular basis of transdetermination have shown that inputs from the Wg, Decapentapelagic (Dpp) and Hedgehog (Hh) signaling pathways activate key selector genes out of their normal developmental context, such as ectopic Vg activation in the leg disc, which then drives cell-fate switches. Several of the genes identified in this screen have functional ties to Wg, Dpp and Hh signaling pathways. For example, Cg is a zinc-finger transcription factor that is required for proper transcriptional regulation of the Hh signaling effector gene Cubitus interruptus (Ci). In cg mutant wing and leg discs, Ci expression is lowered in the anterior compartment, resulting in the ectopic activation of wg and dpp and significant disc overgrowth. Another gene identified in this screen -- ken, functions in concert with Dpp to direct the development of the Drosophila terminalia. Further characterizations of whether these genes and other modifiers of transdetermination and regeneration affect Wg, Dpp and Hh expression and/or signaling may shed light on the regulation of regeneration and regeneration-induced proliferation and cell fate plasticity (McClure, 2008).

GENE STRUCTURE

Genomic length - 7 kb

Exons - 9

PROTEIN STRUCTURE

Amino Acids - 671

Structural Domains

The predicted open
reading frame of LD05357 contains 11 canonical C2H2 zinc-finger
sequences (C-X1-2-C-X3-F-X5-L-X2-H-X3-4-H) typical
of many DNA- and chromatin-binding proteins. A glutamine-rich region (19/24 residues)
spans residues 569 to 592. No significant matches were found to
other proteins outside the zinc-finger and glutamine-rich motifs (Svendsen, 2000 and Campbell, 2000).

combgap encodes a protein with ten putative Krüppel-like C2H2 zinc finger domains. The
amino-terminal region of ~150 amino acids bears little homology to known proteins. The first eight zinc fingers follow in a tandem array, each separated by
6-7 amino acids. Similar clustering of multiple zinc fingers, as well as certain conserved amino acids between and within the zinc fingers, are found in several
predicted human, mouse, and Xenopus multi zinc finger proteins. The presence of a glutamine-rich region flanking the ninth and tenth zinc fingers suggests that
Combgap may act as a DNA binding transcription factor. Consistent with this role, Cg displays nuclear localization (Song, 2000).